Multiple factors in the cancer microenvironment down-regulate tumor immunity and promote tumor progression. In melanoma, one of the dominant mechanisms is the induction of co-inhibitory receptors on tumor-specific T cells and expression of corresponding co-inhibitory ligands by tumor or other cells within tumor stroma, leading to complete or partial loss of T-cell effector functions. Amplified co-inhibitory interactions in the cancer microenvironment impede not only immune tumor surveillance, but also therapeutic immune interventions that may explain the failure of approaches aimed at stimulation of T cells, including tumor antigen-based vaccination and T cell stimulatory cytokines. A prime example of the co-inhibitory interactions is the B7-H1 (PD-L1)/PD-1 pathway. Early studies from our and other laboratories demonstrate a critical role of this pathway in the evasion of cancer immunity, and blockade of this pathway enhances ongoing immune responses against tumors. Recent phase I clinical trials conducted using a fully human antibody to PD-1 demonstrated durable objective (partial and complete) responses in 33% of pretreated metastatic melanoma patients (n=46). Collectively, these studies support B7-H1 as an important co-inhibitory molecule in the down-regulation of immune responses in the melanoma microenvironment and blockade of the B7-H1/PD-1 pathway as one of the most promising approaches for the treatment of melanoma. Project 2 will extend these laboratory and clinical findings in three directions; The specific aims are: Aim 1: to assess the association between B7-H1/PD-1 expression in human melanoma microenvironment with clinical response to anti-PD-1 therapy; Aim 2: to study effector mechanisms of the B7-H1/PD-1 blockade in augmenting anti-melanoma immunity and in melanoma regression; and Aim 3: to maximize melanoma therapeutic immunity by mechanism-based combinatory approaches. Based on our finding that B7-H1 expression by melanoma is directly and extensively up-regulated by interferons, the combination of B7-H1/PD-1 blockade will be tested with IL-2 and interferon-a 2b, and with selective mutant BRAF inhibitors, which promote tumor T-cell infiltration. Our studies should have direct impact for current development of B7-H1/PD-1 blockade as a novel and promising approach for melanoma therapy.